Principles of Plant Genetics and Breeding

and expression (including codon usage, comparing
known sequences, gene searches, etc.).
Identifying and cloning genes of interest
Before a gene can be transferred from one organism
to another, it must be identified and isolated from its
source’s genome. By itself, a piece of DNA cannot selfreplicate.
The isolated gene (or for that matter any DNA
sequence) is maintained in a cell so that copies can be
generated as the cell divides (called cloning).
Cloning vectors
Cloning vectors (or simply vectors) are replicating
units into which isolated fragments of DNA may be
integrated for maintenance. A vector has three basic
features – a selectable marker, a replication origin, and
a cloning site (restriction enzyme site) (Figure 14.1).
Vectors differ in the size of fragments they can stably
incorporate, the procedure for screening the insert
DNA or target DNA, and the number of recombinant
copies they can produce per cell. Examples are:
1 Plasmid vectors. Plasmids are double-stranded, selfreplicating
extrachromosomal molecules with antibiotic
selectable markers. They are small molecules
that can handle small inserts of about 10 kb in size.
RruII
PvuI
PstI
EcoRI
ClaI HindIII
SnaI
BamHI
Tetracycline-resistance gene
Ampicillin-resisitance
gene
PvuII
SphI
Figure 14.1 A cloning vector showing restriction enzyme
sites (no shading) and selection markers sites (dark
shading).
BIOTECHNOLOGY IN PLANT BREEDING 233
SalI
XmaIII
BalI
NruI
AvaI
2 Bacteriophage λ-derived vectors. These are viruses
that infect bacteria. They have a cloning capacity of
about 23 kb.
3 Cosmids. Cosmids are plasmid vectors with cossequences
(for packing) from bacteriophages. They
are capable of carrying about 42 kb inserts.
4 Yeast artificial chromosomes (YAC). These are
useful for cloning large fragments in the megabase
range.
5 Phosphoinositide (PI)-derived artificial chromosome
(PAC) and bacterial artificial chromosome
(BAC). PAC vectors can handle about 350 kb, while
BACs can handle about 150 kb inserts.
Gene isolation and cloning
Gene isolation starts with the construction of a library
representing a set of recombinant molecules that contain
DNA fragments. There are two basic kinds of DNA
libraries (or gene banks) – genomic and cDNA.
Genomic library
DNA is extracted and purified (preferably nuclear DNA).
Fragments of DNA are cloned into appropriate vectors.
The number of recombinant clones needed to create a
gene bank holding the complete genome is given by the
equation:
N = [ln(1 − P)]/[ln(1 − t)n]
where N = number of independent recombinant clones,
P = probability that a particular sequence is represented
(e.g., 95%), t = average length of the fragment to be
cloned (kb), and n = total amount of DNA per cell.
cDNA library
As discussed in Chapter 3, a eukaryotic gene comprises
coding and non-coding segments (exons and introns).
A genomic library contains both elements. A cDNA
library consists of only coding segments of the genome.
This is obtained from the mRNA by reverse transcription.
A cDNA library consists of recombinant molecules
containing all the mRNA in the organism. It is important
to note that only genes that are functionally
expressed in the cell from which the mRNA was isolated
would be included in the library. Consequently, breeders
interested in drought genes, for example, may extract
DNA from a drought-stressed plant, since that condition
is likely to trigger the expression of droughtresistance
genes.